We propose to develop a robust method to encapsulate biological macromolecules inside porous nano-scale (30-100 nm diameter) phospholipid vesicles. The pores will be formed by Staphylococcal toxin alpha-hemolysin. These ultrasmall, biocompatible containers will allow the passage of small molecules such as ATP and magnesium ions, while limiting the diffusional motion of macromolecules inside the zeptoliter volume, thereby enabling new types of biophysical analysis at the single-molecule level. While the method keeps the molecules essentially free of surface artifacts, the vesicles can be tethered to a supported bilayer so that single molecule reactions can be observed for seconds or even minutes. Therefore, this technique has the potential of transforming the way single-molecule fluorescence measurements are performed in many laboratories around the world. We will also pursue light-activatable pores so that biochemical reactions can be triggered locally, which should enable high-throughput, high time resolution single molecule analysis. If the time scale of pore activation is fast, this approach could also prove useful in ensemble kinetic studies since it has a number of advantages over stopped flow methods or conventional uncaging methods. We will use well-characterized systems such as the hairpin ribozyme and the Holliday junction to probe the pore formation process. Furthermore we will use these techniques to make new biological discoveries on the activities of RNA enzymes and helicases.